Methods of Regulating Glucose Metabolism, and Reagents Related Thereto

ABSTRACT

The present invention provides methods and compositions for modification and regulation of glucose and lipid metabolism, generally to reduce insulin resistance, hyperglycemia, hyperinsulinemia, obesity, hyperlipidemia, hyperlipoprotein-emia (such as chylomicrons, VLDL and LDL), and to regulate body fat and more generally lipid stores, and, more generally, for the improvement of metabolism disorders, especially those associated with diabetes, obesity and/or atherosclerosis.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/297,522, filed Nov. 16, 2011, now U.S. Pat. No. 8,513,190; which is acontinuation of U.S. patent application Ser. No. 12/942,313, filed Nov.9, 2010, now U.S. Pat. No. 8,318,669; which is a continuation of U.S.patent application Ser. No. 12/323,751, filed Nov. 26, 2008, now U.S.Pat. No. 7,829,530; which is a continuation of U.S. patent applicationSer. No. 11/487,947, filed Jul. 17, 2006, now U.S. Pat. No. 7,459,428;which is a continuation of U.S. patent application Ser. No. 10/794,316,filed Mar. 4, 2004, now U.S. Pat. No. 7,078,381; which is a continuationof U.S. patent application Ser. No. 10/190,267, filed Jul. 3, 2002, nowU.S. Pat. No. 6,890,898; which is a continuation of U.S. patentapplication Ser. No. 09/628,225, filed Jul. 28, 2000, now U.S. Pat. No.7,157,429; which is a continuation of PCT/US99/02294, filed Feb. 2,1999; which claims the benefit of priority under 35 U.S.C. §119(e) toU.S. Provisional Patent Application Ser. No. 60/073,409, filed Feb. 2,1998.

FUNDING

This invention was made with government support under Grant AI040228awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Diabetes adversely affects the way the body uses sugars and starcheswhich, during digestion, are converted into glucose. Insulin, a hormoneproduced by the pancreas, makes the glucose available to the body'scells for energy. In muscle, adipose (fat) and connective tissues,insulin facilitates the entry of glucose into the cells by an action onthe cell membranes. The ingested glucose is normally converted in theliver to CO₂ and H₂O (50%); to glycogen (5%); and to fat (30-40%), thelatter being stored in fat depots. Fatty acids from the adipose tissuesare circulated, returned to the liver for re-synthesis oftriacylglycerol and metabolized to ketone bodies for utilization by thetissues. The fatty acids are also metabolized by other organs. Fatformation is a major pathway for carbohydrate utilization.

The net effect of insulin is to promote the storage and use ofcarbohydrates, protein and fat. Insulin deficiency is a common andserious pathologic condition in man. In insulin-dependent (IDDM or TypeI) diabetes the pancreas produces little or no insulin, and insulin mustbe injected daily for the survival of the diabetic. Innoninsulin-dependent (NIDDM or Type II) diabetes the pancreas retainsthe ability to produce insulin and in fact may produce higher thannormal amounts of insulin, but the amount of insulin is relativelyinsufficient, or less than fully effective, due to cellular resistanceto insulin.

Diabetes mellitus (DM) is a major chronic illness found in humans withmany consequences. Some complications arising from long-standingdiabetes are blindness, kidney failure, and limb amputations.Insulin-dependent diabetes mellitus (IDDM) accounts for 10 to 15% of allcases of diabetes mellitus. The action of IDDM is to cause hyperglycemia(elevated blood glucose concentration) and a tendency towards diabeticketoacidosis (DKA). Currently treatment requires chronic administrationof insulin. Non-insulin dependent diabetes mellitus (NIDDM) is marked byhyperglycemia that is not linked with DKA. Sporadic or persistentincidence of hyperglycemia can be controlled by administering insulin.Uncontrolled hyperglycemia can damage the cells of the pancreas whichproduce insulin (the β-islet cells) and in the long term create greaterinsulin deficiencies. Currently, oral sulfonylureas and insulin are theonly two therapeutic agents available in the United States for treatmentof diabetes mellitus. Both agents have the potential for producinghypoglycemia as a side effect, reducing the blood glucose concentrationto dangerous levels. There is no generally applicable and consistentlyeffective means of maintaining an essentially normal fluctuation inglucose levels in DM. The resultant treatment attempts to minimize therisks of hypoglycemia while keeping the glucose levels below a targetvalue. The drug regimen is combined with control of dietary intake ofcarbohydrates to keep glucose levels in control.

In either form of diabetes there are widespread abnormalities. In mostNIDDM subjects, the fundamental defects to which the abnormalities canbe traced are (1) a reduced entry of glucose into various “peripheral”tissues and (2) an increased liberation of glucose into the circulationfrom the liver. There is therefore an extracellular glucose excess andan intracellular glucose deficiency. There is also a decrease in theentry of amino acids into muscle and an increase in lipolysis.Hyperlipoproteinemia is also a complication of diabetes. The cumulativeeffect of these diabetes-associated abnormalities is severe blood vesseland nerve damage.

Endocrine secretions of pancreatic islets are regulated by complexcontrol mechanisms driven not only by blood-borne metabolites such asglucose, amino acids, and catecholamines, but also by local paracrineinfluences. Indeed, pancreatic α- and β-cells are critically dependenton hormonal signals generating cyclic AMP (cAMP) as a synergisticmessenger for nutrient-induced hormone release. The major pancreaticislet hormones, glucagon, insulin and somatostatin, interact withspecific pancreatic cell types to modulate the secretory response.Although insulin secretion is predominantly controlled by blood glucoselevels, somatostatin inhibits glucose-mediated insulin secretion.

The human hormone glucagon is a polypeptide hormone produced inpancreatic A-cells. The hormone belongs to a multi-gene family ofstructurally related peptides that include secretin, gastric inhibitorypeptide, vasoactive intestinal peptide and glicentin. These peptidesvariously regulate carbohydrate metabolism, gastrointestinal motilityand secretory processing. However, the principal recognized actions ofpancreatic glucagon are to promote hepatic glycogenolysis andglyconeogenesis, resulting in an elevation of blood sugar levels. Inthis regard, the actions of glucagon are counter regulatory to those ofinsulin and may contribute to the hyperglycemia that accompaniesDiabetes mellitus (Lund et al. (1982) PNAS, 79:345-349).

Preproglucagon, the zymogen form of glucagon, is translated from a 360base pair gene and is processed to form proglucagon (Lund, et al.,supra). Patzelt, et al. (Nature, 282:260-266 (1979)) demonstrated thatproglucagon is further processed into glucagon and a second peptide.Later experiments demonstrated that proglucagon is cleaved carboxyl toLys-Arg or Arg-Arg residues (Lund et al., supra; and Bell et al. (1983)Nature 302:716-718). Bell et al. also discovered that proglucagoncontained three discrete and highly homologous peptide regions whichwere designated glucagon, glucagon-like peptide 1 (GLP-1), andglucagon-like peptide 2 (GLP-2). GLP-1 has attracted increasingattention as a humoral stimulus of insulin secretion. In humans, this29-amino acid peptide, cleaved from proglucagon by cells of theintestinal mucosa, is released into the circulation after nutrientintake (Hoist et al. (1987) FEBS Lett 211:169; Orskov et al. (1987)Diabetologia 30:874; Conlon J (1988) Diabetologia 31:563).

GLP-1 has been found to be a glucose-dependent insulinotropic agent(Gutniak et al. (1992) N Engl. J. Med. 326:1316-1322). GLP-1 is nowknown to stimulate insulin secretion (insulinotropic action) causingglucose uptake by cells which decreases serum glucose levels (see, e.g.,Mojsov, S., Int. J. Peptide Protein Research, 40:333-343 (1992)). Forinstance, it has been shown to be a potent insulin secretagogue inexperimental models and when infused into humans (Gutniak et al., supra;Mojsov et al. (1988) J Clin Invest 79:616; Schmidt et al. (1985)Diabetologia 28:704; and Kreymann et al. (1987) Lancet 2:1300). Thus,GLP-1 is a candidate for the role of an “incretin”, having augmentaryeffects on glucose-mediated insulin release.

It is also noted that numerous GLP-1 analogs have been demonstratedwhich demonstrate insulinotropic action are known in the art. Thesevariants and analogs include, for example, GLP-1(7-36),Gln₉-GLP-1(7-37), D-Gln₉-GLP-1(7-37), acetyl-Lys₉-GLP-1(7-37),Thr₁₆-Lys₁₈-GLP-1(7-37), and Lys₁₈-GLP-1(7-37). Derivatives of GLP-1include, for example, acid addition salts, carboxylate salts, loweralkyl esters, and amides (see, e.g., WO91/11457).

OBJECTS OF THE INVENTION

It is one object of this invention to provide improved methods forreducing in animal subjects (including humans) in need of such treatmentat least one of insulin resistance, hyperinsulinemia, and hyperglycemiaand abating Type II diabetes. Another object is to provide improvedmethods for reducing at least one of body fat stores, hyperlipidemia,hyperlipoproteinemia, and for abating atherosclerosis. It is anotherobject of this invention to provide methods for interfering with glucoseand/or lipid metabolism in a manner beneficial to the host.

It is yet another object of this invention to provide improved methodsfor the long-term reduction and abatement of at least one of theforegoing disorders based on a therapeutic regimen administered over theshort-term.

It is still another object of the present invention to provide a methodfor regulating, and altering on a long term basis, the glucose andlipogenic responses of vertebrate animals, including humans.

In particular, it is an object of the invention to provide methods forproducing long lasting beneficial changes in one or more of thefollowing: the sensitivity of the cellular response of a species toinsulin (reduction of insulin resistance), blood insulin levels,hyperinsulinemia, blood glucose levels, the amount of body fat stores,blood lipoprotein levels, and thus to provide effective treatments fordiabetes, obesity and/or atherosclerosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of the synthesis of a boroproline compound.

FIG. 2 is a glucose tolerance curve which shows that a single injectionof PBP-1 improves glucose levels in blood. The glucose concentration ismeasured before and at 30-minute intervals after the test dose ofglucose. This figure demonstrates that a single injection of PBP-1potentiates the response to a sub-therapeutic dose of GLP-1.

FIG. 3 shows that a single injection of PBP-2 improves glucose levels inblood.

FIG. 4 shows that treatment with PBP-3 under “chronic” conditions alsoresults in lowering of the blood sugar levels.

FIGS. 5A and 5B compare the ability of Pro-boro-pro to lower plasmaglucose levels in GLP-1 receptor−/−transgenic mice.

DETAILED DESCRIPTION OF THE INVENTION

Glucose-induced insulin secretion is modulated by a number of hormonesand neurotransmitters. In particular, two gut hormones, glucagon-likepeptide-1 (GLP-1) and gastric inhibitory peptide (GIP) areinsulinotropic agents, e.g., being agents which can stimulate, or causethe stimulation of, the synthesis or expression of the hormone insulin,are thus called gluco-incretins (Dupre, in The Endocrine Pancreas, E.Samois Ed. (Raven Press, New York, (1991), 253-281); and Ebert et al.(1987) Diabetes Metab. Rev. p3). Glucagon-like peptide-1 is aglucoincretin both in man and other mammals (Dupre et al. supra, andKreymann et al. (1987) Lancet 2:300). It is part of the preproglucagonmolecule (Bell et al. (1983) Nature 304:368) which is proteolyticallyprocessed in intestinal L cells to GLP-1(1-37) and GLP-1(7-36)amide orGLP-1(7-37) (Mojsov et al. (1986) J. Biol. Chem. 261:11880; and Habeneret al.: The Endocrine Pancreas, E. Samois Ed. (Raven Press, New York(1991), 53-71). Only the truncated forms of GLP-1 are biologicallyactive and both have identical effects on insulin secretion in betacells (Mojsov et al. (1987) J. Clin. Invest 79:616; and Weir et al.(1989) Diabetes 38:338). They are the most potent gluco-incretins so fardescribed and are active at concentrations as low as one to tenpicomolar.

The metabolic fate of exogenous GLP-1 has been studied in nondiabeticand type II diabetic subjects. Subcutaneous and intravenous GLP-1 areboth rapidly degraded in a time-dependent manner, for instance, having ahalf-life in diabetic patients of substantially less than 30 minutes.See, for example, Deacon et al. (1995) Diabetes 44:1126-1131.

i. OVERVIEW OF THE INVENTION

The present invention provides methods and compositions for modificationand regulation of glucose and lipid metabolism, generally to reduceinsulin resistance, hyperglycemia, hyperinsulinemia, obesity,hyperlipidemia, hyperlipoproteinemia (such as chylomicrons, VLDL andLDL), and to regulate body fat and more generally lipid stores, and,more generally, for the improvement of metabolism disorders, especiallythose associated with diabetes, obesity and/or atherosclerosis. Asdescribed in greater detail below, the subject method includes theadministration, to an animal, of a composition including one or moredipeptidylpeptidase inhibitors, especially inhibitors of thedipeptidylpeptidase IV (DPIV) enzyme or other enzyme of similarspecificity, which are able to inhibit the proteolysis of GLP-1 andaccordingly increase the plasma half-life of that hormone.

Preferably, the compounds utilized in the subject method will produce anEC50 for the desired biological effect of at least one, two, three andeven four orders of magnitude less than the EC50 for that compound as animmunosuppressant. Indeed, a salient feature of such compounds as thepeptidyl boronates is that the inhibitors can produce, for example, anEC50 for inhibition of glucose tolerance in the nanomolar or less range,whereas the compounds have EC50's for immunosuppression in the μM orgreater range. Thus, a favorable therapeutic index can be realized withrespect to the unwanted side effect of immunosuppression.

While not wishing to bound by any particular theory, it is observed thatcompounds which inhibit DPIV are, correlatively, able to improve glucosetolerance, though not necessarily through mechanisms involving DPIVinhibition per se. Indeed, the results described in Example 6 (and FIG.5) demonstrating an effect in mice lacking a GLP-1 receptor suggest thatthe subject method may not include a mechanism of action directlyimplicating GLP-1 itself, though it has not been ruled out that GLP-1may have other receptors. However, in light of the correlation with DPIVinhibition, in preferred embodiments, the subject method utilizes anagent with a Ki for DPIV inhibition of 1.0 nm or less, more preferablyof 0.1 nm or less, and even more preferably of 0.01 nM or less. Indeed,inhibitors with Ki values in the picomolar and even femtomolar range arecontemplated. Thus, while the active agents are described herein, forconvenience, as “DPIV inhibitors”, it will be understood that suchnomenclature is not intending to limit the subject invention to aparticular mechanism of action.

For instance, in certain embodiments the method involves administrationof a DPIV inhibitor, preferably at a predetermined time(s) during a24-hour period, in an amount effective to improve one or more aberrantindices associated with glucose metabolism disorders (e.g., glucoseintolerance, insulin resistance, hyperglycemia, hyperinsulinemia andType II diabetes).

In other embodiments, the method involves administration of a DPIVinhibitor in an amount effective to improve aberrant indices associatedwith obesity. Fat cells release the hormone leptin, which travels in thebloodstream to the brain and, through leptin receptors there, stimulatesproduction of GLP-1. GLP-1, in turn, produces the sensation of beingfull. The leading theory is that the fat cells of most obese peopleprobably produce enough leptin, but leptin may not be able to properlyengage the leptin receptors in the brain, and so does not stimulateproduction of GLP-1. There is accordingly a great deal of researchtowards utilizing preparations of GLP-1 as an appetite suppressant. Thesubject method provides a means for increasing the half-life of bothendogenous and ectopically added GLP-1 in the treatment of disordersassociated with obesity.

In a more general sense, the present invention provides methods andcompositions for altering the pharmokinetics of a variety of differentpolypeptide hormones by inhibiting the proteolysis of one or morepeptide hormones by DPIV or some other proteolytic activity.Post-secretory metabolism is an important element in the overallhomeostasis of regulatory peptides, and the other enzymes involved inthese processes may be suitable targets for pharmacological interventionby the subject method.

For example, the subject method can be used to increase the half-life ofother proglucagon-derived peptides, such as glicentin (corresponding toPG 1-69), oxyntomodulin (PG 33-69), glicentin-related pancreaticpolypeptide (GRPP, PG 1-30), intervening peptide-2 (IP-2, PG111-122amide), and glucagon-like peptide-2 (GLP-2, PG 126-158).

GLP-2, for example, has been identified as a factor responsible forinducing proliferation of intestinal epithelium. See, for example,Drucker et al. (1996) PNAS 93:7911. The subject method can be used aspart of a regimen for treating injury, inflammation or resection ofintestinal tissue, e.g., where enhanced growth and repair of theintestinal mucosal epithelial is desired.

DPIV has also been implicated in the metabolism and inactivation ofgrowth hormone-releasing factor (GHRF). GHRF is a member of the familyof homologous peptides that includes glucagon, secretin, vasoactiveintestinal peptide (VIP), peptide histidine isoleucine (PHI), pituitaryadenylate cyclase activating peptide (PACAP), gastric inhibitory peptide(GIP) and helodermin. Kubiak et al. (1994) Peptide Res 7:153. GHRF issecreted by the hypothalamus, and stimulates the release of growthhormone (GH) from the anterior pituitary. Thus, the subject method canbe used to improve clinical therapy for certain growth hormone deficientchildren, and in clinical therapy of adults to improve nutrition and toalter body composition (muscle vs. fat). The subject method can also beused in veterinary practice, for example, to develop higher yield milkproduction and higher yield, leaner livestock.

Likewise, the DPIV inhibitors of the subject invention can be used toalter the plasma half-life of secretin, VIP, PHI, PACAP, GIP and/orhelodermin. Additionally, the subject method can be used to alter thepharmacokinetics of Peptide YY and neuropeptide Y, both members of thepancreatic polypeptide family, as DPIV has been implicated in theprocessing of those peptides in a manner which alters receptorselectivity.

Another aspect of the present invention relates to pharmaceuticalcompositions of dipeptidylpeptidase inhibitors, particularly DPIVinhibitors, and their uses in treating and/or preventing disorders whichcan be improved by altering the homeostasis of peptide hormones. In apreferred embodiment, the inhibitors have hypoglycemic and antidiabeticactivities, and can be used in the treatment of disorders marked byaberrant glucose metabolism (including storage). In particularembodiments, the compositions of the subject methods are useful asinsulinotropic agents, or to potentiate the insulinotropic effects ofsuch molecules as GLP-1. In this regard, the present method can beuseful for the treatment and/or prophylaxis of a variety of disorders,including one or more of: hyperlipemia, hyperglycemia, obesity, glucosetolerance insufficiency, insulin resistance and diabetic complications.

In general, the inhibitors of the subject method will be smallmolecules, e.g., with molecular weights less than 7500 amu, preferablyless than 5000 amu, and even more preferably less than 2000 amu and even1000 amu. In preferred embodiments, the inhibitors will be orallyactive.

In certain embodiments, the subject inhibitors are peptidyl compounds(including peptidomimetics) which are optimized, e.g., generally byselection of the Cα substituents, for the substrate specificity of thetargeted proteolytic activity. These peptidyl compounds will include afunctional group, such as in place of the scissile peptide bond, whichfacilitates inhibition of a serine-, cysteine- or aspartate-typeprotease, as appropriate. For example, the inhibitor can be a peptidylα-diketone or a peptidyl α-keto ester, a peptide haloalkylketone, apeptide sulfonyl fluoride, a peptidyl boronate, a peptide epoxide, apeptidyl diazomethanes, a peptidyl phosphonate, isocoumarins,benzoxazin-4-ones, carbamates, isocyanates, isatoic anhydrides or thelike. Such functional groups have been provided in other proteaseinhibitors, and general routes for their synthesis are known. See, forexample, Angelastro et al., J. Med Chem. 33:11-13 (1990); Bey et al.,EPO 363,284; Bey et al., EPO 364,344; Grubb et al., WO 88/10266; Higuchiet al., EPO 393,457; Ewoldt et al., Molecular Immunology 29(6):713-721(1992); Hernandez et al., Journal of Medicinal Chemistry 35(6):1121-1129 (1992); Vlasak et al., J Virology 63(5):2056-2062 (1989);Hudig et al., J Immunol 147(4):1360-1368 (1991); Odakc et al.,Biochemistry 30(8):2217-2227 (1991); Vijayalakshmi et al., Biochemistry30(8):2175-2183 (1991); Kam et al., Thrombosis and Haemostasis64(1):133-137 (1990); Powers et al., J Cell Biochem 39(1):33-46 (1989);Powers et al., Proteinase Inhibitors, Barrett et al., Eds., Elsevier,pp. 55-152 (1986); Powers et al., Biochemistry 29(12):3108-3118 (1990);Oweida et al., Thrombosis Research 58(2):391-397 (1990); Hudig et al.,Molecular Immunology 26(8):793-798 (1989); Orlowski et al., Archives ofBiochemistry and Biophysics 269(1):125-136 (1989); Zunino et al.,Biochimica et Biophysica Acta. 967(3):331-340 (1988); Kam et al.,Biochemistry 27(7):2547-2557 (1988); Parkes et al., Biochem J.230:509-516 (1985); Green et al., J. Biol. Chem. 256:1923-1928 (1981);Angliker et al., Biochem. J. 241:871-875 (1987); Puri et al., Arch.Biochem. Biophys. 27:346-358 (1989); Hanada et al., ProteinaseInhibitors: Medical and Biological Aspects, Katunuma et al., Eds.,Springer-Verlag pp. 25-36 (1983); Kajiwara et al., Biochem. Int.15:935-944 (1987); Rao et al., Thromb. Res. 47:635-637 (1987); Tsujinakaet al., Biochem. Biophys. Res. Commun. 153:1201-1208 (1988)). See alsoU.S. patents Bachovchin et al. U.S. Pat. No. 4,935,493; Bachovchin etal. U.S. Pat. No. 5,462,928; Powers et al. U.S. Pat. No. 5,543,396;Hanko et al. U.S. Pat. No. 5,296,604; and the PCT publication of FerringPCT/GB94/02615.

In other embodiments, the inhibitor is a non-peptidyl compound, e.g.,which can be identified by such drug screening assays as describedherein. These inhibitors can be, merely to illustrate, syntheticorganics, natural products, nucleic acids or carbohydrates.

A representative class of compounds for use in the method of the presentinvention are represented by the general formula;

wherein

A represents a 4-8 membered heterocycle including the N and the Cαcarbon;

Z represents C or N;

W represents a functional group which reacts with an active site residueof the targeted protease, as for example, —CN, —CH═NR₅,

R₁ represents a C-terminally linked amino acid residue or amino acidanalog, or a C-terminally linked peptide or peptide analog, or anamino-protecting group, or

R₂ is absent or represents one or more substitutions to the ring A, eachof which can independently be a halogen, a lower alkyl, a lower alkenyl,a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, ora ketone), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an amino, an acylamino, an amido, a cyano, a nitro, anazido, a sulfate, a sulfonate, a sulfonamido, —(CH₂)_(m)—R₇,—(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, —(CH₂)_(n)—S—(CH₂)_(m)—R₇;

if X is N, R₃ represents hydrogen, if X is C, R₃ represents hydrogen ora halogen, a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl(such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl(such as a thioester, a thioacetate, or a thioformate), an amino, anacylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate,a sulfonamido, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl,—(CH₂)_(m)—O-lower alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl,—(CH₂)_(n)—S—(CH₂)_(m)—R₇;

R₅ represents H, an alkyl, an alkenyl, an alkynyl, —C(X₁)(X₂)X₃,—(CH₂)m-R₇, —(CH₂)n-OH, —(CH₂)n-O-alkyl, —(CH₂)n-O-alkenyl,—(CH₂)n-O-alkynyl, —(CH₂)n-O—(CH₂)m-R₇, —(CH₂)n-SH, —(CH₂)n-S-alkyl,—(CH₂)n-S-alkenyl, —(CH₂)n-S-alkynyl, —(CH₂)n-S—(CH₂)m-R₇, —C(O)C(O)NH₂,—C(O)C(O)OR′₇;

R₆ represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl, anaryl, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl,—(CH₂)_(m)—O-alkenyl, —(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇,—(CH₂)_(m)—SH, —(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl,—(CH₂)_(m)—S-alkynyl, —(CH₂)_(m)—S—(CH₂)_(m)—R₇,

R₇ represents, for each occurrence, a substituted or unsubstituted aryl,aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

R′₇ represents, for each occurrence, hydrogen, or a substituted orunsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl,or heterocycle; and

Y₁ and Y₂ can independently or together be OH, or a group capable ofbeing hydrolyzed to a hydroxyl group, including cyclic derivatives whereY₁ and Y₂ are connected via a ring having from 5 to 8 atoms in the ringstructure (such as pinacol or the like),

R₅₀ represents O or S;

R₅₁ represents N₃, SH₂, NH₂, NO₂ or OR′₇;

R₅₂ represents hydrogen, a lower alkyl, an amine, OR′₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure

X₁ represents a halogen;

X₂ and X₃ each represent a hydrogen or a halogen

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

In preferred embodiments, the ring A is a 5, 6 or 7 membered ring, e.g.,represented by the formula

and more preferably a 5 or 6 membered ring. The ring may, optionally, befurther substituted.

In preferred embodiments, W represents

In preferred embodiments, R1 is

wherein R36 is a small hydrophobic group, e.g., a lower alkyl or ahalogen and R38 is hydrogen, or, R36 and R37 together form a 4-7membered heterocycle including the N and the Cα carbon, as defined for Aabove; and R40 represents a C-terminally linked amino acid residue oramino acid analog, or a C-terminally linked peptide or peptide analog,or an amino-protecting group

In preferred embodiments, R2 is absent, or represents a smallhydrophobic group such as a lower alkyl or a halogen.

In preferred embodiments, R3 is a hydrogen, or a small hydrophobic groupsuch as a lower alkyl or a halogen.

In preferred embodiments, R5 is a hydrogen, or a halogenated loweralkyl.

In preferred embodiments, X1 is a fluorine, and X2 and X3, if halogens,are fluorine.

Also deemed as equivalents are any compounds which can be hydrolyticallyconverted into any of the aforementioned compounds including boronicacid esters and halides, and carbonyl equivalents including acetals,hemiacetals, ketals, and hemiketals, and cyclic dipeptide analogs.

Longer peptide sequences are needed for the inhibition of certainproteases and improve the specificity of the inhibition in some cases.

In preferred embodiments, the subject method utilizes, as a DPIVinhibitor, a boronic acid analogs of an amino acid. For example, thepresent invention contemplates the use of boro-prolyl derivatives in thesubject method. Exemplary boronic acid derived inhibitors of the presentinvention are represented by the general formula:

wherein

R₁ represents a C-terminally linked amino acid residue or amino acidanalog, or a terminally linked peptide or peptide analog, or

C—

R₆ represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl, anaryl, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl,—(CH₂)_(m)—O-alkenyl, —(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇,—(CH₂)_(m)—SH, —(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl,—(CH₂)_(m)—S-alkynyl, —(CH₂)_(m)—S—(CH₂)_(m)—R₇,

R₇ represents an aryl, a cycloalkyl, a cycloalkenyl, or a heterocycle;

R₈ and R₉ each independently represent hydrogen, alkyl, alkenyl,—(CH₂)_(m)—R₇, —C(═O)-alkyl, —C(═O)-alkenyl, —C(═O)-alkynyl,—C(═O)—(CH₂)_(m)—R₇,

or R₈ and R₉ taken together with the N atom to which they are attachedcomplete a heterocyclic ring having from 4 to 8 atoms in the ringstructure;

R₁₁ and R₁₂ each independently represent hydrogen, a alkyl, or apharmaceutically acceptable salt, or R₁₁ and R₁₂ taken together with theO—B—O atoms to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure;

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

In other embodiments, the subject DPIV inhibitors include an aldehydeanalogs of proline or prolyl derivatives. Exemplary aldehyde-derivedinhibitors of the present invention are represented by the generalformula:

wherein

R₁ represents a C-terminally linked amino acid residue or amino acidanalog, or a terminally linked peptide or peptide analog, or

C—

R₆ represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl, anaryl, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl,—(CH₂)_(m)—O-alkenyl, —(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇,—(CH₂)_(m)—SH, —(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl,—(CH₂)_(m)—S-alkynyl, —(CH₂)_(m)—S—(CH₂)_(m)—R₇,

R₇ represents an aryl, a cycloalkyl, a cycloalkenyl, or a heterocycle;

R₈ and R₉ each independently represent hydrogen, alkyl, alkenyl,—(CH₂)_(m)—R₇, —C(═O)-alkyl, —C(═O)-alkenyl, —C(═O)-alkynyl,—C(═O)—(CH₂)_(m)—R₇,

or R₈ and R₉ taken together with the N atom to which they are attachedcomplete a heterocyclic ring having from 4 to 8 atoms in the ringstructure; and

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

In yet further embodiments, the subject DPIV inhibitors are halo-methylketone analogs of an amino acid. Exemplary inhibitors of this classinclude compounds represented by the general formula:

wherein

R₁ represents a C-terminally linked amino acid residue or amino acidanalog, or a terminally linked peptide or peptide analog, or

C—

R₆ represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl, anaryl, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl,—(CH₂)_(m)—O-alkenyl, —(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇,—(CH₂)_(m)—SH, —(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl,—(CH₂)_(m)—S-alkynyl, —(CH₂)_(m)—S—(CH₂)_(m)—R₇,

R₇ represents an aryl, a cycloalkyl, a cycloalkenyl, or a heterocycle;

R₈ and R₉ each independently represent hydrogen, alkyl, alkenyl,—(CH₂)_(m)—R₇, —C(═O)-alkyl, —C(═O)-alkenyl, —C(═O)-alkynyl,—C(═O)—(CH₂)_(m)—R₇,

or R₈ and R₉ taken together with the N atom to which they are attachedcomplete a heterocyclic ring having from 4 to 8 atoms in the ringstructure;

X₁, X₂ and X₃ each represent a hydrogen or a halogen; and

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

In preferred embodiments, the DPIV inhibitor is a peptide orpeptidomimetic including a prolyl group or analog thereof in the P1specificity position, and a nonpolar amino acid in the P2 specificityposition, e.g., a nonpolar amino acid such as alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan or methionine, oran analog thereof. For example, the DPIV inhibitor may include anAla-Pro or Pro-Pro dipeptide sequence or equivalent thereof, and berepresented in the general formulas:

In preferred embodiments, the ring A is a 5, 6 or 7 membered ring, e.g.,represented by the formula

In preferred embodiments, R32 is a small hydrophobic group, e.g., alower alkyl or a halogen.

In preferred embodiments, R30 represents a C-terminally linked aminoacid residue or amino acid analog, or a C-terminally linked peptide orpeptide analog, or an amino-protecting group.

In preferred embodiments, R2 is absent, or represents a smallhydrophobic group such as a lower alkyl or a halogen.

In preferred embodiments, R3 is a hydrogen, or a small hydrophobic groupsuch as a lower alkyl or a halogen.

Another representative class of compounds for use in the subject methodinclude peptide and peptidomimetics of (D)-Ala-(L)-Ala, e.g., preservingthe diastereomeric orientation. Such inhibitors include compoundsrepresented by the general formula:

wherein

W represents a functional group which reacts with an active site residueof the targeted protease, as for example, —CN, —CH═NR₅,

R₁ represents a C-terminally linked amino acid residue or amino acidanalog, or a C-terminally linked peptide or peptide analog, or anamino-protecting group, or

R₃ represents hydrogen or a halogen, a lower alkyl, a lower alkenyl, alower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, or aketone), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an amino, an acylamino, an amido, a cyano, a nitro, anazido, a sulfate, a sulfonate, a sulfonamido, —(CH₂)_(m)—R₇,—(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, —(CH₂)_(n)—S—(CH₂)_(m)—R₇;

R₅ represents H, an alkyl, an alkenyl, an alkynyl, —C(X₁)(X₂)X₃,—(CH₂)m-R₇, —(CH₂)n-OH, —(CH₂)n-O-alkyl, —(CH₂)n-O-alkenyl,—(CH₂)n-O-alkynyl, —(CH₂)n-O—(CH₂)m-R₇, —(CH₂)n-SH, —(CH₂)n-S-alkyl,—(CH₂)n-S-alkenyl, —(CH₂)n-S-alkynyl, —(CH₂)n-S—(CH₂)m-R₇, —C(O)C(O)NH₂,—C(O)C(O)OR′₇;

R₆ represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl, anaryl, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl,—(CH₂)_(m)—O-alkenyl, —(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇,—(CH₂)_(m)—SH, —(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl,—(CH₂)_(m)—S-alkynyl, —(CH₂)_(m)—S—(CH₂)_(m)—R₇,

R₇ represents, for each occurrence, a substituted or unsubstituted aryl,aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

R′₇ represents, for each occurrence, hydrogen, or a substituted orunsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl,or heterocycle;

R₆₁ and R₆₂, independently, represent small hydrophobic groups;

Y₁ and Y₂ can independently or together be OH, or a group capable ofbeing hydrolyzed to a hydroxyl group, including cyclic derivatives whereY₁ and Y₂ are connected via a ring having from 5 to 8 atoms in the ringstructure (such as pinacol or the like),

R₅₀ represents O or S;

R₅₁ represents N₃, SH₂, NH₂, NO₂ or OR′₇;

R₅₂ represents hydrogen, a lower alkyl, an amine, OR′₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure

X₁ represents a halogen;

X₂ and X₃ each represent a hydrogen or a halogen

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

In preferred embodiments, R1 is

wherein R36 is a small hydrophobic group, e.g., a lower alkyl or ahalogen and R38 is hydrogen, or, R36 and R37 together form a 4-7membered heterocycle including the N and the Cα carbon, as defined for Aabove; and R40 represents a C-terminally linked amino acid residue oramino acid analog, or a C-terminally linked peptide or peptide analog,or an amino-protecting group.

In preferred embodiments, R3 is a hydrogen, or a small hydrophobic groupsuch as a lower alkyl or a halogen.

In preferred embodiments, R5 is a hydrogen, or a halogenated loweralkyl.

In preferred embodiments, X1 is a fluorine, and X2 and X3, if halogens,are fluorine.

In preferred embodiments, R₆₁ and R₆₂, independently, represent lowalkyls, such as methyl, ethyl, propyl, isopropyl, tert-butyl or thelike.

Also included are such peptidomimetics as olefins, phosphonates,aza-amino acid analogs and the like.

Also deemed as equivalents are any compounds which can be hydrolyticallyconverted into any of the aforementioned compounds including boronicacid esters and halides, and carbonyl equivalents including acetals,hemiacetals, ketals, and hemiketals, and cyclic dipeptide analogs.

As used herein, the definition of each expression, e.g. alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic salicylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

The pharmaceutically acceptable salts of the present invention can besynthesized from the subject compound which contain a basic or acidmoiety by conventional chemical methods. Generally, the salts areprepared by reacting the free base or acid with stoichiometric amountsor with an excess of the desired salt-forming inorganic or organic acidor base in a suitable solvent. The pharmaceutically acceptable salts ofthe acids of the subject compounds are also readily prepared byconventional procedures such as treating an acid of Formula I with anappropriate amount of a base such as an alkali or alkaline earth methylhydroxide (e.g. sodium, potassium, lithium, calcium or magnesium) or anorganic base such as an amine, piperidine, pyrrolidine, benzylamine andthe like, or a quaternary ammonium hydroxide such as tetramethylammoniumhydroxide and the like.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g. the ability to inhibit proteolysis ofGLP-1 or other peptide hormone or precursor thereof), wherein one ormore simple variations of substituents are made which do not adverselyaffect the efficacy of the compound in use in the contemplated method.In general, the compounds of the present invention may be prepared bythe methods illustrated in the general reaction schemes as, for example,described below, or by modifications thereof, using readily availablestarting materials, reagents and conventional synthesis procedures. Inthese reactions, it is also possible to make use of variants which arein themselves known, but are not mentioned here.

ii. DEFINITIONS

For convenience, before further description of the present invention,certain terms employed in the specification, examples, and appendedclaims are collected here.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ for branchedchain), and more preferably 20 or fewer. Likewise, preferred cycloalkylshave from 3-10 carbon atoms in their ring structure, and more preferablyhave 5, 6 or 7 carbons in the ring structure.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification and claims is intended to include both “unsubstitutedalkyls” and “substituted alkyls”, the latter of which refers to alkylmoieties having substituents replacing a hydrogen on one or more carbonsof the hydrocarbon backbone. Such substituents can include, for example,a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an ester, aformyl, or a ketone), a thiocarbonyl (such as a thioester, athioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphonate,a phosphinate, an amino, an amido, an amidine, an imine, a cyano, anitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, asulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or anaromatic or heteroaromatic moiety. It will be understood by thoseskilled in the art that the moieties substituted on the hydrocarbonchain can themselves be substituted, if appropriate. For instance, thesubstituents of a substituted alkyl may include substituted andunsubstituted forms of amino, azido, imino, amido, phosphoryl (includingphosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido,sulfamoyl and sulfonate), and silyl groups, as well as ethers,alkylthios, carbonyls (including ketones, aldehydes, carboxylates, andesters), —CF₃, —CN and the like. Exemplary substituted alkyls aredescribed below. Cycloalkyls can be further substituted with alkyls,alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls,—CF₃, —CN, and the like.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Preferred alkyl groups are lower alkyls. Inpreferred embodiments, a substituent designated herein as alkyl is alower alkyl.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromaticmoieties, —CF₃, —CN, or the like. The term “aryl” also includespolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings (the rings are “fusedrings”) wherein at least one of the rings is aromatic, e.g., the othercyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,sulfur and phosphorous.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The terms “amine” and “amino” are art recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R₈, or R₉ and R₁₀ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R₈ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In preferred embodiments, only one of R₉ or R₁₀can be a carbonyl, e.g., R₉, R₁₀ and the nitrogen together do not forman imide. In even more preferred embodiments, R₉ and R₁₀ (and optionallyR′₁₀) each independently represent a hydrogen, an alkyl, an alkenyl, or—(CH₂)_(m)—R₈. Thus, the term “alkylamine” as used herein means an aminegroup, as defined above, having a substituted or unsubstituted alkylattached thereto, i.e., at least one of R₉ and R₁₀ is an alkyl group.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′₁₁ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “carbonyl” is art recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thiolester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₈,where m and R₈ are described above.

The term “sulfonate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that canbe represented by the general formula:

in which R₉ and R′₁₁ are as defined above.

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₉ and R₁₀ are as defined above.

The terms “sulfoxido” or “sulfinyl”, as used herein, refers to a moietythat can be represented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

A “phosphoryl” can in general be represented by the formula:

wherein Q₁ represented S or O, and R₄₆ represents hydrogen, a loweralkyl or an aryl. When used to substitute, e.g., an alkyl, thephosphoryl group of the phosphorylalkyl can be represented by thegeneral formula:

wherein Q₁ represented S or O, and each R₄₆ independently representshydrogen, a lower alkyl or an aryl, Q₂ represents O, S or N. When Q₁ isan S, the phosphoryl moiety is a “phosphorothioate”.

A “phosphoramidite” can be represented in the general formula:

wherein R₉ and R₁₀ are as defined above, and Q₂ represents O, S or N.

A “phosphonamidite” can be represented in the general formula:

wherein R₉ and R₁₀ are as defined above, Q₂ represents O, S or N, andR₄₈ represents a lower alkyl or an aryl, Q₂ represents O, S or N.

A “selenoalkyl” refers to an alkyl group having a substituted selenogroup attached thereto. Exemplary “selenoethers” which may besubstituted on the alkyl are selected from one of —Se-alkyl,—Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R₇, m and R₇ being definedabove.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described hereinabove. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalencies of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

A “small” substituent is one of 10 atoms or less.

By the terms “amino acid residue” and “peptide residue” is meant anamino acid or peptide molecule without the —OH of its carboxyl group. Ingeneral the abbreviations used herein for designating the amino acidsand the protective groups are based on recommendations of the IUPAC-IUBCommission on Biochemical Nomenclature (see Biochemistry (1972)11:1726-1732). For instance Met, Ile, Leu, Ala and Gly represent“residues” of methionine, isoleucine, leucine, alanine and glycine,respectively. By the residue is meant a radical derived from thecorresponding α-amino acid by eliminating the OH portion of the carboxylgroup and the H portion of the α-amino group. The term “amino acid sidechain” is that part of an amino acid exclusive of the —CH(NH₂)COOHportion, as defined by K. D. Kopple, “Peptides and Amino Acids”, W. A.Benjamin Inc., New York and Amsterdam, 1966, pages 2 and 33; examples ofsuch side chains of the common amino acids are —CH₂CH₂SCH₃ (the sidechain of methionine), —CH₂(CH₃)—CH₂CH₃ (the side chain of isoleucine),—CH₂CH(CH₃)₂ (the side chain of leucine) or H-(the side chain ofglycine).

For the most part, the amino acids used in the application of thisinvention are those naturally occurring amino acids found in proteins,or the naturally occurring anabolic or catabolic products of such aminoacids which contain amino and carboxyl groups. Particularly suitableamino acid side chains include side chains selected from those of thefollowing amino acids: glycine, alanine, valine, cysteine, leucine,isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid,glutamine, asparagine, lysine, arginine, proline, histidine,phenylalanine, tyrosine, and tryptophan, and those amino acids and aminoacid analogs which have been identified as constituents ofpeptidylglycan bacterial cell walls.

The term amino acid residue further includes analogs, derivatives andcongeners of any specific amino acid referred to herein, as well asC-terminal or N-terminal protected amino acid derivatives (e.g. modifiedwith an N-terminal or C-terminal protecting group). For example, thepresent invention contemplates the use of amino acid analogs wherein aside chain is lengthened or shortened while still providing a carboxyl,amino or other reactive precursor functional group for cyclization, aswell as amino acid analogs having variant side chains with appropriatefunctional groups). For instance, the subject compound can include anamino acid analog such as, for example, cyanoalanine, canavanine,djenkolic acid, norleucine, 3-phosphoserine, homoserine,dihydroxy-phenylalanine, 5-hydroxytryptophan, 1-methylhistidine,3-methylhistidine, diaminopimelic acid, ornithine, or diaminobutyricacid. Other naturally occurring amino acid metabolites or precursorshaving side chains which are suitable herein will be recognized by thoseskilled in the art and are included in the scope of the presentinvention.

Also included are the (D) and (L) stereoisomers of such amino acids whenthe structure of the amino acid admits of stereoisomeric forms. Theconfiguration of the amino acids and amino acid residues herein aredesignated by the appropriate symbols (D), (L) or (DL), furthermore whenthe configuration is not designated the amino acid or residue can havethe configuration (D), (L) or (DL). It will be noted that the structureof some of the compounds of this invention includes asymmetric carbonatoms. It is to be understood accordingly that the isomers arising fromsuch asymmetry are included within the scope of this invention. Suchisomers can be obtained in substantially pure form by classicalseparation techniques and by sterically controlled synthesis. For thepurposes of this application, unless expressly noted to the contrary, anamed amino acid shall be construed to include both the (D) or (L)stereoisomers.

The phrase “protecting group” as used herein means substituents whichprotect the reactive functional group from undesirable chemicalreactions. Examples of such protecting groups include esters ofcarboxylic acids and boronic acids, ethers of alcohols and acetals andketals of aldehydes and ketones. For instance, the phrase “N-terminalprotecting group” or “amino-protecting group” as used herein refers tovarious amino-protecting groups which can be employed to protect theN-terminus of an amino acid or peptide against undesirable reactionsduring synthetic procedures. Examples of suitable groups include acylprotecting groups such as, to illustrate, formyl, dansyl, acetyl,benzoyl, trifluoroacetyl, succinyl and methoxysuccinyl; aromaticurethane protecting groups as, for example, benzyloxycarbonyl (Cbz); andaliphatic urethane protecting groups such as t-butoxycarbonyl (Boc) or9-Fluorenylmethoxycarbonyl (FMOC).

As noted above, certain compounds of the present invention may exist inparticular geometric or stereoisomeric forms. The present inventioncontemplates all such compounds, including cis- and trans-isomers, R-and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term “hydrocarbon” is contemplatedto include all permissible compounds having at least one hydrogen andone carbon atom. In a broad aspect, the permissible hydrocarbons includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

A compound is said to have an “insulinotropic activity” if it is able tostimulate, or cause the stimulation of, the synthesis or expression ofthe hormone insulin.

iii. EXEMPLARY FORMULATIONS

A. Agonism of GLP-1 Effects

The inhibitors useful in the subject methods possess, in certainembodiments, the ability to lower blood glucose levels, to relieveobesity, to alleviate impaired glucose tolerance, to inhibit hepaticglucose neogenesis, and to lower blood lipid levels and to inhibitaldose reductase. They are thus useful for the prevention and/or therapyof hyperglycemia, obesity, hyperlipidemia, diabetic complications(including retinopathy, nephropathy, neuropathy, cataracts, coronaryartery disease and arteriosclerosis) and furthermore for obesity-relatedhypertension and osteoporosis.

Diabetes mellitus is a disease characterized by hyperglycemia occurringfrom a relative or absolute decrease in insulin secretion, decreasedinsulin sensitivity or insulin resistance. The morbidity and mortalityof this disease result from vascular, renal, and neurologicalcomplications. An oral glucose tolerance test is a clinical test used todiagnose diabetes. In an oral glucose tolerance test, a patient'sphysiological response to a glucose load or challenge is evaluated.After ingesting the glucose, the patient's physiological response to theglucose challenge is evaluated. Generally, this is accomplished bydetermining the patient's blood glucose levels (the concentration ofglucose in the patient's plasma, serum or whole blood) for severalpredetermined points in time.

As described in the appended examples, we demonstrate that, in vivo,high affinity inhibitors of DPIV are biologically active with respect toregulation of glucose metabolism. For example, a single injection of theinhibitor Pro-boro-Pro (see examples for structure) was alone sufficientto improve glucose control. A single injection of Pro-boro-Pro was alsoobserved to potentiate the response to a subtherapeutic dose of GLP-1.We have also observed that chronic (>5 days) treatment with Pro-boro-Proalone lowers both fasting blood sugars, and the glycemic excursion tooral glucose challenge.

As indicated above, the inhibitors useful in the subject method can bepeptide- or peptidomimetic-derived inhibitors of the target proteolyticactivity, or can be a non-peptide compound identified, e.g., by drugscreening assays described herein. With respect to DPIV inhibitors, asalient feature of the subject method is the unexpected finding thatcertain DPIV inhibitors have antidiabetic activity at concentrationssignificantly lower than the EC50 of the compound as animmunosuppressant. Thus, an animal can be dosed under a regimen designedto provide a blood serum concentration of inhibitor at or about the EC50for antidiabetic effects, and still be sufficiently below the EC50 forimmunosuppressive activity so as to avoid complications resulting fromthat activity. Indeed, for certain of the subject inhibitors, it isanticipated that dosing can be at least an order of magnitude or moregreater than the antidiabetic EC50, yet still remain sufficiently belowa dose producing any significant immunosuppression.

As discussed further below, a variety of assays are available in the artfor identifying potential inhibitors of DPIV and the like, as well asassessing the various biological activities (including side-effects andtoxicity) of such an inhibitor.

B. Agonism of Other Peptide Hormones

In another embodiment, the subject agents can be used to agonize (e.g.,mimic or potentiate) the activity of other polypeptide hormones.

To illustrate, the present invention provides a method for agonizing theaction of GLP-2. It has been determined that GLP-2 acts as a trophicagent, to promote growth of gastrointestinal tissue. The effect of GLP-2is marked particularly by increased growth of the small bowel, and istherefore herein referred to as an “intestinotrophic” effect.

In still other embodiments, the subject method can be used to increasethe half-life of other proglucagon-derived peptides, such as glicentin,oxyntomodulin, glicentin-related pancreatic polypeptide (GRPP), and/orintervening peptide-2 (IP-2). For example, glicentin has beendemonstrated to cause proliferation of intestinal mucosa and alsoinhibits a peristalsis of the stomach, and has thus been elucidated asuseful as a therapeutic agent for digestive tract diseases, thus leadingto the present invention.

Thus, in one aspect, the present invention relates to therapeutic andrelated uses of DPIV inhibitors for promoting the growth andproliferation of gastrointestinal tissue, most particularly small boweltissue. For instance, the subject method can be used as part of aregimen for treating injury, inflammation or resection of intestinaltissue, e.g., where enhanced growth and repair of the intestinal mucosalepithelial is desired.

With respect to small bowel tissue, such growth is measured convenientlyas a increase in small bowel mass and length, relative to an untreatedcontrol. The effect of subject inhibitors on small bowel also manifestsas an increase in the height of the crypt plus villus axis. Suchactivity is referred to herein as an “intestinotrophic” activity. Theefficacy of the subject method may also be detectable as an increase incrypt cell proliferation and/or a decrease in small bowel epitheliumapoptosis. These cellular effects may be noted most significantly inrelation to the jejunum, including the distal jejunum and particularlythe proximal jejunum, and also in the distal ileum. A compound isconsidered to have “intestinotrophic effect” if a test animal exhibitssignificantly increased small bowel weight, increased height of thecrypt plus villus axis, or increased crypt cell proliferation ordecreased small bowel epithelium apoptosis when treated with thecompound (or genetically engineered to express it themselves). A modelsuitable for determining such gastrointestinal growth is described byU.S. Pat. No. 5,834,428.

In general, patients who would benefit from either increased smallintestinal mass and consequent increased small bowel mucosal functionare candidates for treatment by the subject method. Particularconditions that may be treated include the various forms of sprueincluding celiac sprue which results from a toxic reaction to α-gliadinfrom wheat, and is marked by a tremendous loss of villae of the bowel;tropical sprue which results from infection and is marked by partialflattening of the villae; hypogammaglobulinemic sprue which is observedcommonly in patients with common variable immunodeficiency orhypogammaglobulinemia and is marked by significant decrease in villusheight. The therapeutic efficacy of the treatment may be monitored byenteric biopsy to examine the villus morphology, by biochemicalassessment of nutrient absorption, by patient weight gain, or byamelioration of the symptoms associated with these conditions. Otherconditions that may be treated by the subject method, or for which thesubject method may be useful prophylactically, include radiationenteritis, infectious or post-infectious enteritis, regional enteritis(Crohn's disease), small intestinal damage due to toxic or otherchemotherapeutic agents, and patients with short bowel syndrome.

More generally, the present invention provides a therapeutic method fortreating digestive tract diseases. The term “digestive tract” as usedherein means a tube through which food passes, including stomach andintestine. The term “digestive tract diseases” as used herein meansdiseases accompanied by a qualitative or quantitative abnormality in thedigestive tract mucosa, which include, e.g., ulceric or inflammatorydisease; congenital or acquired digestion and absorption disorderincluding malabsorption syndrome; disease caused by loss of a mucosalbarrier function of the gut; and protein-losing gastroenteropathy. Theulceric disease includes, e.g., gastric ulcer, duodenal ulcer, smallintestinal ulcer, colonic ulcer and rectal ulcer. The inflammatorydisease include, e.g., esophagitis, gastritis, duodenitis, enteritis,colitis, Crohn's disease, proctitis, gastrointestinal Behcet, radiationenteritis, radiation colitis, radiation proctitis, enteritis andmedicamentosa. The malabsorption syndrome includes the essentialmalabsorption syndrome such as disaccharide-decomposing enzymedeficiency, glucose-galactose malabsorption, fractose malabsorption;secondary malabsorption syndrome, e.g., the disorder caused by a mucosalatrophy in the digestive tract through the intravenous or parenteralnutrition or elemental diet, the disease caused by the resection andshunt of the small intestine such as short gut syndrome, cul-de-sacsyndrome; and indigestible malabsorption syndrome such as the diseasecaused by resection of the stomach, e.g., dumping syndrome.

The term “therapeutic agent for digestive tract diseases” as used hereinmeans the agents for the prevention and treatment of the digestive tractdiseases, which include, e.g., the therapeutic agent for digestive tractulcer, the therapeutic agent for inflammatory digestive tract disease,the therapeutic agent for mucosal atrophy in the digestive tract and thetherapeutic agent for digestive tract wound, the amelioration agent forthe function of the digestive tract including the agent for recovery ofthe mucosal barrier function and the amelioration agent for digestiveand absorptive function. The ulcers include digestive ulcers anderosions, acute ulcers, namely, acute mucosal lesions.

The subject method, because of promoting proliferation of intestinalmucosa, can be used in the treatment and prevention of pathologicconditions of insufficiency in digestion and absorption, that is,treatment and prevention of mucosal atrophy, or treatment of hypoplasiaof the digestive tract tissues and decrease in these tissues by surgicalremoval as well as improvement of digestion and absorption. Further, thesubject method can be used in the treatment of pathologic mucosalconditions due to inflammatory diseases such as enteritis, Crohn'sdisease and ulceric colitis and also in the treatment of reduction infunction of the digestive tract after operation, for example, in dampingsyndrome as well as in the treatment of duodenal ulcer in conjunctionwith the inhibition of peristalsis of the stomach and rapid migration offood from the stomach to the jejunum. Furthermore, glicentin caneffectively be used in promoting cure of surgical invasion as well as inimproving functions of the digestive tract. Thus, the present inventionalso provides a therapeutic agent for atrophy of the digestive tractmucosa, a therapeutic agent for wounds in the digestive tract and a drugfor improving functions of the digestive tract which comprise glicentinas active ingredients.

Likewise, the DPIV inhibitors of the subject invention can be used toalter the plasma half-life of secretin, VIP, PHI, PACAP, GIP and/orhelodermin. Additionally, the subject method can be used to alter thepharmacokinetics of Peptide YY and neuropeptide Y, both members of thepancreatic polypeptide family, as DPIV has been implicated in theprocessing of those peptides in a manner which alters receptorselectivity.

Neuropeptide Y (NPY) is believed to act in the regulation vascularsmooth muscle tone, as well as regulation of blood pressure. NPY alsodecreases cardiac contractility. NPY is also the most powerful appetitestimulant known (Wilding et al., (1992) J Endocrinology 132:299-302).The centrally evoked food intake (appetite stimulation) effect ispredominantly mediated by NPY Y1 receptors and causes increase in bodyfat stores and obesity (Stanley et al., (1989) Physiology and Behavior46:173-177).

According to the present invention, a method for treatment of anorexiacomprises administering to a host subject an effective amount of a DPIVinhibitor to stimulate the appetite and increase body fat stores whichthereby substantially relieves the symptoms of anorexia.

A method for treatment of hypotension comprises administering to a hostsubject an effective amount of a DPIV inhibitor of the present inventionto mediate vasoconstriction and increase blood pressure which therebysubstantially relieves the symptoms of hypotension.

DPIV has also been implicated in the metabolism and inactivation ofgrowth hormone-releasing factor (GHRF). GHRF is a member of the familyof homologous peptides that includes glucagon, secretin, vasoactiveintestinal peptide (VIP), peptide histidine isoleucine (PHI), pituitaryadenylate cyclase activating peptide (PACAP), gastric inhibitory peptide(GIP) and helodermin. Kubiak et al. (1994) Peptide Res 7:153. GHRF issecreted by the hypothalamus, and stimulates the release of growthhormone (GH) from the anterior pituitary. Thus, the subject method canbe used to improve clinical therapy for certain growth hormone deficientchildren, and in clinical therapy of adults to improve nutrition and toalter body composition (muscle vs. fat). The subject method can also beused in veterinary practice, for example, to develop higher yield milkproduction and higher yield, leaner livestock.

C. Examples of Peptidyl DPIV Inhibitors

In the case of DPIV inhibitors, a preferred class of inhibitors arepeptidyl compounds based on the dipeptides Pro-Pro or Ala-Pro. Anotherpreferred class of peptidyl inhibitors are compounds based on thedipeptide (D)-Ala-(L)-Ala. In many embodiments, it will be desirable toprovide the peptidyl moiety as a peptidomimetic, e.g., to increasebioavailability and/or increase the serum half-life relative to theequivalent peptide. For instance, a variety of peptide backbone analogsare available in the art and be readily adapted for use in the subjectmethods.

In an exemplary embodiment, the peptidomimetic can be derived as aretro-inverso analog of the peptide. To illustrate, certain of thesubject peptides can be generated as the retro-inverso analog (shown inits unprotected state):

Such retro-inverso analogs can be made according to the methods known inthe art, such as that described by the Sisto et al. U.S. Pat. No.4,522,752. For example, the illustrated retro-inverso analog can begenerated as follows. The geminal diamine corresponding to theN-terminal amino acid analogs is synthesized by treating anN-Boc-protected amino acid (having the sidechain R) with ammonia underHOBT-DCC coupling conditions to yield amide, and then effecting aHofmann-type rearrangement with I,I-bis-(trifluoroacetoxy)iodobenzene(TIB), as described in Radhakrishna et al. (1979) J. Org. Chem. 44:1746.The product amine salt is then coupled to a side-chain protected (e.g.,as the benzyl ester) N-Fmoc D-enantiomer of the second amino acidresidue (e.g., having a sidechain R′) under standard conditions to yieldthe pseudodipeptide. The Fmoc (fluorenylmethoxycarbonyl) group isremoved with piperidine in dimethylformamide, and the resulting amine istrimethylsilylated with bistrimethylsilylacetamide (BSA) beforecondensation with suitably alkylated, side-chain protected derivative ofMeldrum's acid, as described in U.S. Pat. No. 5,061,811 to Pinori etal., to yield the retro-inverso tripeptide analog. The pseudotripeptideis then coupled with (protected) boro-proline under standard conditionsto give the protected tetrapeptide analog. The protecting groups areremoved to release the final product, which is purified by HPLC.

In another illustrative embodiment, the peptidomimetic can be derived asa retro-enantio analog of the peptide.

Retro-enantio analogs such as this can be synthesized usingD-enantiomers of commercially available D-amino acids or other aminoacid analogs and standard solid- or solution-phase peptide-synthesistechniques.

In still another illustrative embodiment, trans-olefin derivatives canbe made with the subject boronophenylalanine analogs. For example, anexemplary olefin analog is:

The trans olefin analog can be synthesized according to the method of Y.K. Shue et al. (1987) Tetrahedron Letters 28:3225.

Still another class of peptidomimetic boronophenylalanine derivativesinclude the phosphonate derivatives, such as:

The synthesis of such phosphonate derivatives can be adapted from knownsynthesis schemes. See, for example, Loots et al. in Peptides: Chemistryand Biology, (Escom Science Publishers, Leiden, 1988, p. 118); Petrilloet al. in Peptides: Structure and Function (Proceedings of the 9thAmerican Peptide Symposium, Pierce Chemical Co. Rockland, Ill., 1985).

D. Non-Peptidyl DPIV Inhibitors

The pharmaceutical industry has developed a variety of differentstrategies for assessing millions of compounds a year as potential leadcompounds based on inhibitory activity against an enzyme. DPIV and otherproteolytic enzymes targeted by the subject method are amenable to thetypes of high throughput screening required to sample large arrays ofcompounds and natural extracts for suitable inhibitors.

As an illustrative embodiment, the ability of a test agent to inhibitDPIV can be assessed using a colorimetric or fluorometric substrate,such as Ala-Pro-paranitroanilide. See U.S. Pat. No. 5,462,928. Moreover,DPIV can be purified, and is accordingly readily amenable for use insuch high throughput formats as multi-well plates.

Briefly, DPIV is purified from pig kidney cortex (Barth et al. (1974)Acta Biol Med Germ 32:157; Wolf et al. (1972) Acta Bio Mes Germ 37:409)or human placenta (Puschel et al. (1982) Eur J Biochem 126:359). Anillustrative reaction mixture includes 50 μM sodium Hepes (pH7.8), 10 μMAla-Pro-paranitroanilide, 6 milliunits of DPIV, and 2% (v/v)dimethylformamide in a total volume of 1.0 mL. The reaction is initiatedby addition of enzyme, and formation of reaction product(paranitroanilide) in the presence and absence of a test compound can bedetected photometrically, e.g., at 410 nm.

Exemplary compounds which can be screened for activity against DPIV (orother relevant enzymes) include peptides, nucleic acids, carbohydrates,small organic molecules, and natural product extract libraries, such asisolated from animals, plants, fungus and/or microbes.

E. Assays of Insulinotropic Activity

In selecting a compound suitable for use in the subject method, it isnoted that the insulinotropic property of a compound may be determinedby providing that compound to animal cells, or injecting that compoundinto animals and monitoring the release of immunoreactive insulin (IRI)into the media or circulatory system of the animal, respectively. Thepresence of IRI can be detected through the use of a radioimmunoassaywhich can specifically detect insulin.

The db/db mouse is a genetically obese and diabetic strain of mouse. Thedb/db mouse develops hyperglycemia and hyperinsulinemia concomitant withits development of obesity and thus serves as a model of obese type 2diabetes (NIDDM). The db/db mice can purchased from, for example, TheJackson Laboratories (Bar Harbor, Me.). In an exemplary embodiment, fortreatment of the mice with a regimen including a DPIV inhibitor orcontrol, sub-orbital sinus blood samples are taken before and at sometime (e.g., 60 minutes) after dosing of each animal. Blood glucosemeasurements can be made by any of several conventional techniques, suchas using a glucose meter. The blood glucose levels of the control andDPIV inhibitor dosed animals are compared

The metabolic fate of exogenous GLP-1 can also be followed in eithernondiabetic and type II diabetic subjects, and the effect of a candidateDPIV inhibitor determined. For instance, a combination of high-pressureliquid chromatography (HPLC), specific radioimmunoassays (RIAs), and aenzyme-linked immunosorbent assay (ELISA), can be used, whereby intactbiologically active GLP-1 and its metabolites can be detected. See, forexample, Deacon et al. (1995) Diabetes 44:1126-1131. To illustrate,after GLP-1 administration, the intact peptide can be measured using anNH2-terminally directed RIA or ELISA, while the difference inconcentration between these assays and a COOH-terminal-specific RIAallowed determination of NH2-terminally truncated metabolites. Withoutinhibitor, subcutaneous GLP-1 is rapidly degraded in a time-dependentmanner, forming a metabolite which co-elutes on HPLC with GLP-I(9-36)amide and has the same immunoreactive profile. For instance, thirtyminutes after subcutaneous GLP-1 administration to diabetic patients(n=8), the metabolite accounted for 88.5+1.9% of the increase in plasmaimmunoreactivity determined by the COOH-terminal RIA, which was higherthan the levels measured in healthy subjects (78.4+3.2%; n=8; P<0.05).See Deacon et al., supra. Intravenously infused GLP-I was alsoextensively degraded.

F. Pharmaceutical Formulations

The inhibitors can be administered in various forms, depending on thedisorder to be treated and the age, condition and body weight of thepatient, as is well known in the art. For example, where the compoundsare to be administered orally, they may be formulated as tablets,capsules, granules, powders or syrups; or for parenteral administration,they may be formulated as injections (intravenous, intramuscular orsubcutaneous), drop infusion preparations or suppositories. Forapplication by the ophthalmic mucous membrane route, they may beformulated as eyedrops or eye ointments. These formulations can beprepared by conventional means, and, if desired, the active ingredientmay be mixed with any conventional additive, such as an excipient, abinder, a disintegrating agent, a lubricant, a corrigent, a solubilizingagent, a suspension aid, an emulsifying agent or a coating agent.Although the dosage will vary depending on the symptoms, age and bodyweight of the patient, the nature and severity of the disorder to betreated or prevented, the route of administration and the form of thedrug, in general, a daily dosage of from 0.01 to 2000 mg of the compoundis recommended for an adult human patient, and this may be administeredin a single dose or in divided doses.

Glucose metabolism can be altered, and symptoms associated with type IIdiabetes can be decreased or eliminated, in accordance with a “timed”administration of DPIV inhibitors wherein one or more appropriateindices for glucose metabolism and/or type II diabetes can be used toassess effectiveness of the treatment (dosage and/or timing): e.g.glucose tolerance, glucose level, insulin level, insulin sensitivity,glycosylated hemoglobin.

An effective time for administering DPIV inhibitors needs to beidentified. This can be accomplished by routine experiment as describedbelow, using one or more groups of animals (preferably at least 5animals per group).

In animals, insulinotropic activity by DPIV inhibitor treatment can beassessed by administering the inhibitor at a particular time of day andmeasuring the effect of the administration (if any) by measuring one ormore indices associated with glucose metabolism, and comparing thepost-treatment values of these indices to the values of the same indicesprior to treatment.

The precise time of administration and/or amount of DPIV inhibitor thatwill yield the most effective results in terms of efficacy of treatmentin a given patient will depend upon the activity, pharmacokinetics, andbioavailability of a particular compound, physiological condition of thepatient (including age, sex, disease type and stage, general physicalcondition, responsiveness to a given dosage and type of medication),route of administration, etc. However, the above guidelines can be usedas the basis for fine-tuning the treatment, e.g., determining theoptimum time and/or amount of administration, which will require no morethan routine experimentation consisting of monitoring the subject andadjusting the dosage and/or timing.

While the subject is being treated, glucose metabolism is monitored bymeasuring one or more of the relevant indices at predetermined timesduring a 24-hour period. Treatment (amounts, times of administration andtype of medication) may be adjusted (optimized) according to the resultsof such monitoring. The patient is periodically reevaluated to determineextent of improvement by measuring the same parameters, the first suchreevaluation typically occurring at the end of four weeks from the onsetof therapy, and subsequent reevaluations occurring every 4 to 8 weeksduring therapy and then every 3 months thereafter. Therapy may continuefor several months or even years with six months being a typical lengthof therapy for humans.

Adjustments to the amount(s) of drug(s) administered and possibly to thetime of administration may be made based on these reevaluations. Forexample, if after 4 weeks of treatment one of the metabolic indices hasnot improved but at least one other one has, the dose could be increasedby ⅓ without changing the time of administration.

Treatment can be initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage should be increasedby small increments until the optimum effect under the circumstances isreached. For convenience, the total daily dosage may be divided andadministered in portions during the day if desired.

The phrase “therapeutically-effective amount” as used herein means thatamount of, e.g., a DPIV inhibitor(s), which is effective for producingsome desired therapeutic effect by inhibiting, for example, theproteolysis of a peptide hormone at a reasonable benefit/risk ratioapplicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose DPIV inhibitors, materials, compositions, and/or dosage formswhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject chemical fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

The term “pharmaceutically-acceptable salts” refers to the relativelynon-toxic, inorganic and organic acid addition salts of DPIV inhibitors.These salts can be prepared in situ during the final isolation andpurification of the DPIV inhibitors, or by separately reacting apurified DPIV inhibitor in its free base form with a suitable organic orinorganic acid, and isolating the salt thus formed. Representative saltsinclude the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate,nitrate, acetate, valerate, oleate, palmitate, stearate, laurate,benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate,and laurylsulphonate salts and the like. (See, for example, Berge et al.(1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19)

In other cases, the DPIV inhibitor useful in the methods of the presentinvention may contain one or more acidic functional groups and, thus,are capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable bases. The term “pharmaceutically-acceptablesalts” in these instances refers to the relatively non-toxic, inorganicand organic base addition salts of a DPIV inhibitor(s). These salts canlikewise be prepared in situ during the final isolation and purificationof the DPIV inhibitor(s), or by separately reacting the purified DPIVinhibitor(s) in its free acid form with a suitable base, such as thehydroxide, carbonate or bicarbonate of a pharmaceutically-acceptablemetal cation, with ammonia, or with a pharmaceutically-acceptableorganic primary, secondary or tertiary amine Representative alkali oralkaline earth salts include the lithium, sodium, potassium, calcium,magnesium, and aluminum salts and the like. Representative organicamines useful for the formation of base addition salts includeethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine,piperazine and the like (see, for example, Berge et al., supra).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations useful in the methods of the present invention includethose suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal, aerosol and/or parenteral administration.The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a DPIV inhibitor(s) with the carrier and,optionally, one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation a DPIV inhibitor with liquid carriers, or finely dividedsolid carriers, or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of a DPIV inhibitor(s) as an active ingredient. Acompound may also be administered as a bolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically-acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, acetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered peptide orpeptidomimetic moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active ingredient, the liquid dosage formsmay contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active DPIV inhibitor(s) may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more DPIVinhibitor(s) with one or more suitable nonirritating excipients orcarriers comprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of a DPIVinhibitor(s) include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active componentmay be mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to DPIVinhibitor(s), excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to a DPIV inhibitor(s),excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

The DPIV inhibitor(s) can be alternatively administered by aerosol. Thisis accomplished by preparing an aqueous aerosol, liposomal preparationor solid particles containing the compound. A nonaqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers arepreferred because they minimize exposing the agent to shear, which canresult in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of a DPIV inhibitor(s) to the body. Such dosage forms can bemade by dissolving or dispersing the agent in the proper medium.Absorption enhancers can also be used to increase the flux of thepeptidomimetic across the skin. The rate of such flux can be controlledby either providing a rate controlling membrane or dispersing thepeptidomimetic in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more DPIV inhibitor(s) in combinationwith one or more pharmaceutically-acceptable sterile isotonic aqueous ornonaqueous solutions, dispersions, suspensions or emulsions, or sterilepowders which may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofDPIV inhibitor(s) in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the DPIV inhibitor(s) of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of agents may be given orally, parenterally, topically,or rectally. They are of course given by forms suitable for eachadministration route. For example, they are administered in tablets orcapsule form, by injection, inhalation, eye lotion, ointment,suppository, etc. administration by injection, infusion or inhalation;topical by lotion or ointment; and rectal by suppositories. Oraladministration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a DPIV inhibitor, drug or othermaterial other than directly into the central nervous system, such thatit enters the patient's system and, thus, is subject to metabolism andother like processes, for example, subcutaneous administration.

These DPIV inhibitor(s) may be administered to humans and other animalsfor therapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the DPIVinhibitor(s), which may be used in a suitable hydrated form, and/or thepharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

G. Conjoint Administration

Another aspect of the invention provides a conjoint therapy wherein oneor more other therapeutic agents are administered with the proteaseinhibitor. Such conjoint treatment may be achieved by way of thesimultaneous, sequential or separate dosing of the individual componentsof the treatment.

In one embodiment, a DPIV inhibitor is conjointly administered withinsulin or other insulinotropic agents, such as GLP-1 or a gene therapyvector which causes the ectopic expression of GLP-1.

In another illustrative embodiment, the subject inhibitors can beconjointly administered with a an M1 receptor antagonist. Cholinergicagents are potent modulators of insulin release that act via muscarinicreceptors. Moreover, the use of such agents can have the added benefitof decreasing cholesterol levels, while increasing HDL levels. Suitablemuscarinic receptor antagonists include substances that directly orindirectly block activation of muscarinic cholinergic receptors.Preferably, such substances are selective (or are used in amounts thatpromote such selectivity) for the M1 receptor. Nonlimiting examplesinclude quaternary amines (such as methantheline, ipratropium, andpropantheline), tertiary amines (e.g. dicyclomine, scopolamine) andtricyclic amines (e.g. telenzepine). Pirenzepine and methyl scopolamineare preferred. Other suitable muscarinic receptor antagonists includebenztropine (commercially available as COGENTIN from Merck),hexahydro-sila-difenidol hydrochloride (HHSID hydrochloride disclosed inLambrecht et al. (1989) Trends in Pharmacol. Sci. 10(Suppl):60;(+/−)-3-quinuclidinyl xanthene-9-carboxylate hemioxalate(QNX-hemioxalate; Birdsall et al., Trends in Pharmacol. Sci. 4:459,1983; telenzepine dihydrochloride (Coruzzi et al. (1989) Arch. Int.Pharmacodyn. Ther. 302:232; and Kawashima et al. (1990) Gen. Pharmacol.21:17) and atropine. The dosages of such muscarinic receptor antagonistswill be generally subject to optimization as outlined above. In the caseof lipid metabolism disorders, dosage optimization may be necessaryindependently of whether administration is timed by reference to thelipid metabolism responsiveness window or not.

In terms of regulating insulin and lipid metabolism and reducing theforegoing disorders, the subject DPIV inhibitors may also actsynergistically with prolactin inhibitors such as d2 dopamine agonists(e.g. bromocriptine). Accordingly, the subject method can include theconjoint administration of such prolactin inhibitors asprolactin-inhibiting ergo alkaloids and prolactin-inhibiting dopamineagonists. Examples of suitable compounds include2-bromo-alpha-ergocriptine, 6-methyl-8beta-carbobenzyloxyaminoethyl-10-alpha-ergoline, 8-acylaminoergolines,6-methyl-8-alpha-(N-acyl)amino-9-ergoline,6-methyl-8-alpha-(N-phenylacetyl)amino-9-ergoline, ergocornine,9,10-dihydroergocornine, D-2-halo-6-alkyl-8-substituted ergolines,D-2-bromo-6-methyl-8-cyanomethylergoline, carbidopa, benserazide andother dopadecarboxylase inhibitors, L-dopa, dopamine and non toxic saltsthereof.

The DPIV inhibitors used according to the invention can also be usedconjointly with agents acting on the ATP-dependent potassium channel ofthe β-cells, such as glibenclamide, glipizide, gliclazide and AG-EE 623ZW. The DPIV inhibitors may also advantageously be applied incombination with other oral agents such as metformin and relatedcompounds or glucosidase inhibitors as, for example, acarbose.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Synthesis of BoroProline

Referring to FIG. 1, the starting compound I is prepared essentially bythe procedure of Matteson et al. (Organometallics 3:1284, 1984), exceptthat a pinacol ester is substituted for the pinanediol ester. Similarcompounds such as boropipecolic acid and 2-azetodine boronic acid can beprepared by making the appropriate selection of starting material toyield the pentyl and propyl analogs of compound I. Further, Cl can besubstituted for Br in the formula, and other diol protecting groups canbe substituted for pinacol in the formula, e.g., 2,3-butanediol andalphapinanediol.

Compound II is prepared by reacting compound I with [(CH₃)O₃Si]₂N—Li⁺.In this reaction hexamethyldisilazane is dissolved in tetrahydrofuranand an equivalent of n-butyllithium added at −78° C. After warming toroom temperature (20° C.) and cooling to −78° C., an equivalent ofcompound I is added in tetrahydrofuran. The mixture is allowed to slowlycome to room temperature and to stir overnight. Thealpha-bis[trimethylsilane]-protected amine is isolated by evaporatingsolvent and adding hexane under anhydrous conditions. Insoluble residueis removed by filtration under a nitrogen blanket, yielding a hexanesolution of compound II.

Compound III, the N-trimethysilyl protected form of boroProline isobtained by the thermal cyclization of compound II during thedistillation process in which compound II is heated to 100-150° C. anddistillate is collected which boils 66-62° C. at 0.06-0.10 mm pressure.

Compound IV, boroProline-pinacol hydrogen chloride, is obtained bytreatment of compound III with HCl:dioxane. Excess HCl and by-productsare removed by trituration with ether. The final product is obtained ina high degree of purity by recrystallization from ethyl acetate.

The boroProline esters can also be obtained by treatment of the reactionmixture obtained in the preparation of compound II with anhydrous acidto yield 1-amino-4-bromobutyl boronate pinacol as a salt. Cyclizationoccurs after neutralizing the salt with base and heating the reaction.

Example 2 Preparation of BoroProline Pinacol

The intermediate, 4-Bromo-1-chlorobutyl boronate pinacol, was preparedby the method in Matteson et al. (Organometallics 3:1284, 1984) exceptthat conditions were modified for large scale preparations and pinacolwas substituted for the pinanediol protecting group.

3-bromopropyl boronate pinacol was prepared by hydrogenboronation ofallyl bromide (173 ml, 2.00 moles) with catechol borane (240 ml, 2.00moles). Catechol borane was added to allyl bromide and the reactionheated for 4 hours at 100° C. under a nitrogen atmosphere. The product,3-bromopropyl boronate catechol (bp 95-102° C., 0.25 mm), was isolatedin a yield of 49% by distillation. The catechol ester (124 g, 0.52moles) was transesterified with pinacol (61.5 g, 0.52 moles) by mixingthe component in 50 ml of THF and allowing them to stir for 0.5 hours at0° C. and 0.5 hours at room temperature. Solvent was removed byevaporation and 250 ml of hexane added. Catechol was removed as acrystalline solid. Quantitative removal was achieved by successivedilution to 500 ml and to 1000 ml with hexane and removing crystals ateach dilution. Hexane was evaporated and the product distilled to yield177 g (bp 60-64° C., 0.35 mm).

4-Bromo-1-chlorobutyl boronate pinacol was prepared by homologation ofthe corresponding propyl boronate. Methylene chloride (50.54 ml, 0.713moles) was dissolved in 500 ml of THF, 1.54N n-butyllithium in hexane(480 ml, 0.780 moles) was slowly added at −100° C. 3-Bromopropylboronate pinacol (178 g, 0.713 moles) was dissolved in 500 ml of THG,cooled to the freezing point of the solution, and added to the reactionmixture. Zinc chloride (54.4 g, 0.392 moles) was dissolved in 250 ml ofTHG, cooled to 0° C., and added to the reaction mixture in severalportions. The reaction was allowed to slowly warm to room temperatureand to stir overnight. Solvent was evaporated and the residue dissolvedin hexane (1 liter) and washed with water (1 liter). Insoluble materialwas discarded. After drying over anhydrous magnesium sulfate andfiltering, solvent was evaporated. The product was distilled to yield147 g (bp 110-112° C., 0.200 mm)

N-Trimethylsilyl-boroProline pinacol was prepared first by dissolvinghexamethyldisilizane (20.0 g, 80.0 mmoles) in 30 ml of THF, cooling thesolution to −78° C., and adding 1.62N n-butyllithium in hexane (49.4 ml,80.0 mmoles). The solution was allowed to slowly warm to roomtemperature. It was recooled to −78° C. and 4-bromo-1-chlorobutylboronate pinacol (23.9 g, 80.0 mmoles) added in 20 ml of THF. Themixture was allowed to slowly warm to room temperature and to stirovernight. Solvent was removed by evaporation and dry hexane (400 ml)added to yield a precipitant which was removed by filtration under anitrogen atmosphere. The filtrate was evaporated and the residuedistilled, yielding 19.4 g of the desired product (bp 60-62° C.,0.1-0.06 mm)

H-boroProline-pinacol.HCl (boroProline-pinacol.HCl) was prepared bycooling N-trimethylsilyl-boroProline pinacol (16.0 g, 61.7 mmoles) to−78° C. and adding 4N HCL:dioxane 46 ml, 185 mmoles). The mixture wasstirred 30 minutes at −78° C. and 1 hour at room temperature. Solventwas evaporated and the residue triturated with ether to yield a solid.The crude product was dissolved in chloroform and insoluble materialremoved by filtration. The solution was evaporated and the productcrystallized from ethyl acetate to yield 11.1 g of the desired product(mp 156.5-157° C.).

Example 3 Synthesis of BoroProline Peptides

General methods of coupling of N-protected peptides and amino acids withsuitable side-chain protecting groups to H-boroProline-pinacol areapplicable. When needed, side-chain protecting and N-terminal protectinggroups can be removed by treatment with anhydrous HCl, HBr,trifluoroacetic acid, or by catalytic hydrogenation. These proceduresare known to those skilled in the art of peptide synthesis.

The mixed anhydride procedure of Anderson et al. (J. Am. Chem. Soc.89:5012, 1984) is preferred for peptide coupling. Referring again toFIG. 1, the mixed anhydride of an N-protected amino acid or a peptide isprepared by dissolving the peptide in tetrahydrofuran and adding oneequivalent of N-methylmorpholine. The solution is cooled to −20° C. andan equivalent of isobutyl chloroformate is added. After 5 minutes, thismixture and one equivalent of triethylamine (or other stericallyhindered base) are added to a solution of H-boroPro-pinacol dissolved ineither cold chloroform of tetrahydrofuran.

The reaction mixture is routinely stirred for one hour at −20° C. and 1to 2 hours at room temperature (20° C.). Solvent is removed byevaporation, and the residue is dissolved in ethyl acetate. The organicsolution is washed with 0.20N hydrochloric acid, 5% aqueous sodiumbicarbonate, and saturated aqueous sodium chloride. The organic phase isdried over anhydrous sodium sulfate, filtered, and evaporated. Productsare purified by either silica gel chromatography or gel permeationchromatography using Sephadex TM LH-20 and methanol as a solvent.

Previous studies have shown that the pinacol protecting group can beremoved in situ by preincubation in phosphate buffer prior to runningbiological experiments (Kettner et al., J. Biol. Chem. 259:15106, 1984).Several other methods are also applicable for removing pinacol groupsfrom peptides, including boroProline, and characterizing the finalproduct. First, the peptide can be treated with diethanolamine to yieldthe corresponding diethanolamine boronic acid ester, which can bereadily hydrolyzed by treatment with aqueous acid or a sulfonic acidsubstituted polystyrene resin as described in Kettner et al. (supra).Both pinacol and pinanediol protecting groups can be removed by treatingwith BC13 in methylene chloride as described by Kinder et al. (J. Med.Chem. 28:1917). Finally, the free boronic acid can be converted to thedifluoroboron derivative (—BF2) by treatment with aqueous HF asdescribed by Kinder et al. (supra).

Similarly, different ester groups can be introduced by reacting the freeboronic acid with various di-hydroxy compounds (for example, thosecontaining heteroatoms such as S or N) in an inert solvent.

Example 4 Preparation of H-Ala-boroPro

Boc-Ala-boroPro was prepared by mixed anhydride coupling of theN-Boc-protected alanine and H-boroPro prepared as described above.H-Ala-boroPro (Ala-boroPro) was prepared by removal of the Bocprotecting group at 0° C. in 3.5 molar excess of 4N HCl-dioxane. Thecoupling and deblocking reactions were performed by standard chemicalreaction. Ala-boroPro has a K_(i) for DP-IV of in the nanomolar range.Boc-blocked Ala-boroPro has no affinity for DP-IV.

The two diastereomers of Ala-boroPro-pinacol, L-Ala-D-boroPro-pinacoland L-Ala-L-boroPro-pinacol, can be partially separated by silica gelchromatography with 20% methanol in ethyl acetate as eluant. The earlyfraction appears by NMR analysis to be 95% enriched in one isomer.Because this fraction has more inhibits DP-IV to a greater extent thanlater fractions (at equal concentrations) it is probably enriched in theL-boroPro (L-Ala-L-boroPro-pinacol) isomer.

Example 5 Glucose Tolerance Test

Experiments show that Pro-boro-pro clearly lowers blood sugar based uponresults from an oral glucose challenge in mice. The first twoexperiments are “acute” experiments wherein the mice were injected witha single dose of Pro-boro-pro. In the first set of experiments mice wereinjected with 150 μg of Pro-boro-pro (PBP-1) and then subjected to anoral glucose tolerance test within an hour. 8 μg of GLP-1 was alsoadministered to these mice five minutes prior to administration ofglucose. See FIG. 2. In a second set of experiments mice were injectedwith Pro-boro-pro (PBP-2) one hour prior to an oral glucose challengetest. FIG. 3 presents the results of these experiments. Each set ofexperiments was also performed using saline as a control.

The third set of experiments were “chronic” experiments, wherein themice were injected twice daily with Pro-boro-pro for four days, followedby an oral glucose challenge. These results are presented in FIG. 4.

Example 6 Glucose Tolerance Test, Comparison of Normal and GLP-1Receptor−/−Mice

GLP-1 receptor gene “knock-out” causes glucose intolerance in transgenicmice. Gallwitz B; Schmidt WE Z Gastroenterol (1997) 35: 655-8. FIG. 5compares the ability of Pro-boro-pro to lower plasma glucose levels innormal and GLP-1 receptor−/−transgenic mice.

All of the above-cited references and publications are herebyincorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-37. (canceled)
 38. A method for modifying glucose metabolism,comprising administering orally to an animal in need thereof atherapeutically effective amount of an inhibitor of dipeptidylpeptidaseIV (DPIV) or a pharmaceutically acceptable salt thereof once daily,wherein the inhibitor has a Ki for inhibition of DPIV of 10 nM or less;the duration of the therapeutic effect is at least about 24 hours; andthe inhibitor is administered in an amount sufficient to increase theplasma half-life of glucagon-like peptide 1 (GLP-1) but not sufficientto suppress the immune system of the animal.
 39. The method of claim 38,wherein the inhibitor has a Ki for inhibition of DPIV of 1.0 nM or less.40. The method of claim 39, wherein the inhibitor has a Ki forinhibition of DPIV of 0.1 nM or less.
 41. The method of claim 40,wherein the inhibitor has a Ki for inhibition of DPIV of 0.01 nM orless.
 42. The method of claim 38, wherein the inhibitor has an EC₅₀ fortreatment of Type II diabetes at least one order of magnitude less thanits EC₅₀ for immunosuppression.
 43. The method of claim 42, wherein theinhibitor has an EC₅₀ for treatment of Type II diabetes at least twoorders of magnitude less than its EC₅₀ for immunosuppression.
 44. Themethod of claim 38, wherein the inhibitor has a molecular weight lessthan 5000 amu.
 45. The method of claim 44, wherein the inhibitor has amolecular weight less than 2000 amu.
 46. The method of claim 45, whereinthe inhibitor has a molecular weight less than 1000 amu.
 47. The methodof claim 38, wherein the animal is a mammal.
 48. The method of claim 47,wherein the mammal is a human.
 49. The method of claim 38, wherein theinhibitor is administered in a solid dosage form.
 50. The method ofclaim 49, wherein the solid dosage form is a tablet, capsule or pill.51. The method of claim 49, wherein the solid dosage form is a tablet.52. The method of claim 51, wherein the solid dosage form is a coatedtablet.
 53. The method of claim 38, wherein the duration of thetherapeutic effect is about 24 hours.
 54. The method of claim 38,wherein the animal is a human; and the duration of the therapeuticeffect is about 24 hours.
 55. The method of claim 38, wherein the animalis a human; and the inhibitor has a molecular weight less than 1000 amu.56. The method of claim 38, wherein the animal is a human; and theinhibitor is administered in the form of a tablet, capsule or pill. 57.The method of claim 38, wherein the animal is a human; and the inhibitoris administered in the form of a tablet.
 58. The method of claim 38,wherein the animal is a human; and the inhibitor is administered in theform of a coated tablet.
 59. The method of claim 38, wherein the animalis a human; the inhibitor is administered in the form of a tablet,capsule or pill; and the inhibitor has a molecular weight less than 1000amu.
 60. The method of claim 38, wherein the animal is a human; theinhibitor is administered in the form of a tablet; and the inhibitor hasa molecular weight less than 1000 amu.
 61. The method of claim 38,wherein the animal is a human; the inhibitor is administered in the formof a coated tablet; and the inhibitor has a molecular weight less than1000 amu.
 62. The method of claim 38, wherein the animal is a human; theinhibitor is administered in the form of a tablet, capsule or pill; andthe duration of the therapeutic effect is about 24 hours.
 63. The methodof claim 38, wherein the animal is a human; the inhibitor isadministered in the form of a tablet; and the duration of thetherapeutic effect is about 24 hours.
 64. The method of claim 38,wherein the animal is a human; the inhibitor is administered in the formof a coated tablet; and the duration of the therapeutic effect is about24 hours.
 65. The method of claim 38, wherein the animal is a human; theinhibitor is administered in the form of a tablet, capsule or pill; theduration of the therapeutic effect is about 24 hours; and the inhibitorhas a molecular weight less than 1000 amu.
 66. The method of claim 38,wherein the animal is a human; the inhibitor is administered in the formof a tablet; the duration of the therapeutic effect is about 24 hours;and the inhibitor has a molecular weight less than 1000 amu.
 67. Themethod of claim 38, wherein the animal is a human; the inhibitor isadministered in the form of a coated tablet; the duration of thetherapeutic effect is about 24 hours; and the inhibitor has a molecularweight less than 1000 amu.